Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith

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OriginalspracheEnglisch
Seiten (von - bis)474-489
Seitenumfang16
FachzeitschriftApplied Biochemistry and Biotechnology
Jahrgang178
Ausgabenummer3
PublikationsstatusVeröffentlicht - 13 Okt. 2015

Abstract

The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5 %) and β-caryophyllene (~5.5 %) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters KM and kcat were determined using a discontinuous kinetic assay.

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Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith. / Alemdar, Semra; Hartwig, Steffen; Frister, Thore et al.
in: Applied Biochemistry and Biotechnology, Jahrgang 178, Nr. 3, 13.10.2015, S. 474-489.

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

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title = "Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith",
abstract = "The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5 %) and β-caryophyllene (~5.5 %) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters KM and kcat were determined using a discontinuous kinetic assay.",
keywords = "Enzyme activity, Humulene, Purification, Recombinant expression, Sesquiterpene, Terpene synthase",
author = "Semra Alemdar and Steffen Hartwig and Thore Frister and K{\"o}nig, {Jan Christoph} and Thomas Scheper and Sascha Beutel",
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journal = "Applied Biochemistry and Biotechnology",
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T1 - Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith

AU - Alemdar, Semra

AU - Hartwig, Steffen

AU - Frister, Thore

AU - König, Jan Christoph

AU - Scheper, Thomas

AU - Beutel, Sascha

PY - 2015/10/13

Y1 - 2015/10/13

N2 - The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5 %) and β-caryophyllene (~5.5 %) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters KM and kcat were determined using a discontinuous kinetic assay.

AB - The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5 %) and β-caryophyllene (~5.5 %) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters KM and kcat were determined using a discontinuous kinetic assay.

KW - Enzyme activity

KW - Humulene

KW - Purification

KW - Recombinant expression

KW - Sesquiterpene

KW - Terpene synthase

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U2 - 10.1007/s12010-015-1888-4

DO - 10.1007/s12010-015-1888-4

M3 - Article

C2 - 26463657

AN - SCOPUS:84959518981

VL - 178

SP - 474

EP - 489

JO - Applied Biochemistry and Biotechnology

JF - Applied Biochemistry and Biotechnology

SN - 0273-2289

IS - 3

ER -

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